1. Field of the Invention
The present invention relates to the field of automated analyzers for nucleic acid diagnostics, in particular to devices which will contain the sample and reagents during thermal cycling.
2. Description of the Related Art
It is well known in the field of molecular biology that a reaction is influenced by the temperature at which the reaction is performed. If the temperature of the reaction varies, the results could be inconsistent with previous assays or with results of the calibration reactions. A device which provides heating and cooling of sample and reagents without evaporative loss is useful in many processes and particularly useful in gene amplification and detection processes. A device open to surrounding ambient air loses water vapor from aqueous fluids and, without adding water to replenish the water vapor loss, solute concentrations increase as rising temperatures drive more of the aqueous solvent from the device. It is well known in the field of molecular biology that a reaction is influenced by the concentrations of solutes in which the reaction is performed. It is also known that diffusion in and out of cells is affected by pressure. Air and vapor pressure within a sealed device increase as air and water are heated.
The device of this invention forms at least one closed chamber over a standard microscope slide or a carrier base, for example, the carrier described in U.S. Pat. application No. 5,188,963 issued Feb. 23, 1993, and pending U.S. Pat. applications Ser. No. 07/836,348 and Ser. No. 07/855,318now U.S. Pat. No. 5,281,516 issued Jan. 25, 1994. The carrier in the aforementioned references comprises a unidirectional fluid flow channel, defined by a carrier base and a top cover portion, through which reaction fluids are introduced for complexation with target molecules of a specimen fixed to the carrier base, with the provision of a collection trough into which spent fluids drain from the carrier base.
The device of this invention may be used alone, or integrated with the carrier base supporting multiple slides, so that each chamber encloses a specimen area on the upper surface of the slides or the carrier base containing specimen holding areas. The device of this invention describes a top cover portion comprising individual chambers to be used when an evaporation-proof chamber is desired. The specimen holding area between the slide, or carrier base, and the cover are sealed for precise temperature regulation without evaporative loss of reaction fluids. Said device is interchangeable with the top cover portion as designed previously for sequential addition of a series of reagents and washes for detection.
The in situ amplification process (described in U.S. Pat. No. 5,188,963 and co-pending U.S. Pat. applications, Ser. No. 935,637, filed Aug. 24, 1992, which is a continuation of U.S. Pat. application Ser. No. 07/227,348, filed Aug. 2, 1988, now abandoned; Ser. No. 07/836,348, filed Mar. 3, 1992; and Ser. No. 07/929,720, filed Aug. 12, 1992) uses enzymes such as polymerase or ligase, separately or in combination, to repeatedly generate more copies of a target nucleic acid sequence within cells by primer extensions to incorporate new nucleotides or by ligations of adjacent complementary oligonucleotides, wherein each template generates more copies and the copies may themselves become template. By melting complementary strands of nucleic acids, the original strand and each new strand synthesized are potential templates for repeated primer annealing or ligation reactions to make and expand the number of specific, amplified products. A thermostable polymerase with reverse transcriptase activity and a thermostable ligase are now both available and increase the choice of enzymes and combination of reactions for in situ applications. If RNA in the sample is the target to be amplified, the sample is treated with reverse transcriptase to make a nucleic acid complement of the RNA just prior to amplification. Using a thermostable reverse transcriptase polymerase such as rTth (Perkin Elmer, Norwalk, Conn.), it may not be necessary to add another polymerase for rounds of primer extension amplification. The amplification can either be primer extensions in one direction for linear amplification, or in opposing directions, for geometric amplification. The label can either be incorporated as labeled nucleotides or labeled primers for one-step detection or labeled probes may be added in a step following amplification whereby the probes hybridize to the amplified products for detection.
Until in situ amplification was invented, nucleic acid amplification was limited to solution reactions wherein the nucleic acid is released from cells or tissue prior to amplification of the target sequence. In the aforementioned co-pending patent applications, a process to amplify nucleic acid targets within cells was invented and a method for embedding the cellular specimens in a matrix was described to immobilize and stabilize the cells during amplification and detection. A number of examples for using in situ amplification are given in application Ser. No. 7/438,592 now U.S. Pat. No. 5,188,963 issued on Feb. 23, 1993. A photomicrograph of cells which had amplified, labeled DNA was included in co-pending application Ser. No. 7/836,348 to show that the amplified fragments are retained in individual cells and such cells can be enumerated under microscopic observation,
The process requires at least one denaturing or high temperature stage, and one primer annealing or low temperature stage in each cycle. To achieve the desired results, as stated in Ser. No. 07/836,348, the embedded cell samples are heated to nucleic acid denaturation temperature and temperature control commences before reagent addition. Since the specificity of binding one nucleic acid oligonucleotide or strand to a complementary nucleic acid is influenced by temperature, uniform and accurate temperature from sample to sample is needed for the reaction. The time required for the sample to be brought to the reaction temperatures can be a large percentage of the time allowed for the biochemical processes to be performed; therefore, means to cycle the temperature of small volumes of reagents rapidly and reliably are desirable.
It is commonly known to seal or cement cover slips to slides in order to preserve a specimen. The current techniques to prevent evaporation of reagents at elevated temperatures consist of covering the specimen, such as cells or tissue fixed on a microscope slide, with a cover slip and sealing the cover slip with either rubber cement, lacquers known commonly as finger nail polish or similar adhesives and/or overlaying the specimen with mineral oil as a vapor barrier. In the former case the adhesive bonds must be broken to remove the cover and in the latter case the mineral oil must be removed in order to continue further processing for detection. It is known in the art to use chloroform or acetone to soften hardened nail polish. These techniques are unsatisfactory because they are messy, labor-intensive and introduce unwanted material and additional steps.
Once a chamber is made with the slide or carrier base, means of adding reagents within it are necessary. Means of adding solutions to closed systems, which we have tried, include injection ports made of natural rubber (such as injection sites #75-32 from Abbott Laboratories, Ashland, Ohio), wherein reagents are injected with a standard hypodermic needle or the like. A rubber gum injection port is made so the gum material closes over the opening made by the needle when the needle is removed. These injection ports are not adaptable for automated fluid delivery probes as they require significant force to pierce. Furthermore methods using needles should be limited as handling them introduces risks to personnel.
Applying reagents with a standard pipette tip or the like is more advantageous. Flexible tubing was clamped or crimped to seal after pipetting reagents through the tubing, but sealing a small chamber with tools has difficulties. If covers are thin-walled, an injection port is as simple as making an opening in the cover by piercing the cover with a sharp point of a fluid-delivery device and then covering the opening with adhesives known in the art such as mylar tape. Mylar tape was sufficient in our experience to maintain liquids in a chamber repeatedly heated and cooled when the cover was flexible enough to vent vapor pressure. Piercing is a crude method and, since the opening must be large enough for air to escape, the reagents may leak out the opening when it is being sealed. Double bore tubing or needles may be used in which one lumen is much smaller than the other, the smaller lumen being the means by which air escapes from the chamber as fluid reagents are introduced into the chamber. The device of the invention improves on all of these possibilities by sealing the chamber, providing simple means of introducing reagents into the chamber and reclosing while also providing means to vent pressure.
Lab-Tek.RTM. culture chamber slides (Nunc Inc., Naperyills, Ill.) are used to add growth media and an inoculum of cells for incubation in separate chambers on a microscope slide. After incubation the chamber may be separated from the slide by removing the chamber, lid and gasket in order to easily stain the cells adhering to the slide. Each Lab-Tek.RTM. culture cheer slide is covered by setting a lid over the chamber. Ordinary air pressure and gravity provide a sufficient seal during incubations at 30-37.degree. C. A large air space relative to the volume of media is present above the media. If such a chamber were to be used for rapid temperature cycling reactions to temperatures near 95.degree. C., water molecules evaporating in the air space above the liquid would exert a pressure on the lid whereby the water molecules would escape the chamber or condense on the lid whose temperature is cooler than the water vapor, said water vapor removed from the reaction liquids by hanging as water droplets from the under surface of a lid. Loss of water molecules in the reaction mix with the specimen reduces or stops enzymatic activity.
The aforementioned in situ amplification for cellular analyses, which requires precise temperature regulation, creates a need for an improved apparatus which controls the temperature of the cellular specimens and prevents evaporative loss during thermal cycling. A device designed to make a reaction chamber over slide specimens such that the chamber is sealed sufficiently to prevent evaporative loss during thermal cycling, but which may be easily removed or converted to a chamber in which subsequent reagents may be added and washed away from the specimen is desirable. Thin, flat devices containing an ultra-thin specimen on its upper surface, which bottom surface makes contact with heating means, transfers and spreads the heat quickly to the specimen.
The device of this invention is a cover placed over the specimen area of the slide forming a chamber into which reagents are either placed before sealing to a slide or preferably filled after the covers are sealed to the slides. The chamber has a thin configuration over the specimen holding area to minimize the volume of reagents needed to saturate a thin specimen. Specimens are prepared to be thin for convenient microscopic analysis. The device of this invention enables and does not interfere with subsequent microscopic detection of signals within the immobilized cells or tissue specimen.
Patent applications which describe automated gene detection instrumentation and in situ sample preparation, in situ amplification and in situ detection are U.S. Pat. application Ser. No. 07/438,592, now U.S. Pat. No. 5,188,963 issued on Feb. 23, 1993 which is a continuation of U.S. Pat. application Ser. No. 07/227,348 filed Aug. 2, 1988 and now abandoned, and co-pending U.S. Pat. application Ser. No. 07/836,348, filed Mar. 3, 1992, which is a national phase application based on international patent application Ser. No. PCT/US90/06768, which has a priority date of Nov. 17, 1989, based on U.S. Pat. application Ser. No. 07/438,592, issued as U.S. Pat. No. 5,188,963 on Feb. 23, 1993; and U.S. application Ser. No. 07/855,318 filed Mar. 23, 1992 and now U.S. Pat. No. 5,281,516 issued on Jan. 25, 1994, the disclosures of all of which are incorporated herein by reference. An object of the invention is to provide a chamber for detecting molecular targets in a thin biological specimen at temperatures elevated above ambient temperature.
A further object of the invention is to temporarily seal said chamber against a discrete area of a thin specimen fixed onto a microscope slide or onto an optically clear flat base.
A yet further object of the invention is to provide means for adding and removing reaction fluids to said chamber.
A yet further object of the invention is to provide means to minimize evaporative loss of reaction fluids from said chamber.
A yet further object of the invention is to provide means to minimize pressure increases within the chamber which result form heating the chamber.
A yet further object of the invention is to either enhance a uniform refluence of water vapor back into the reaction fluids or minimize such a refluence to prevent partitioning of fluids within the chamber.
Other aspects and features of the invention will more fully apparent from a consideration of the following disclosure and appended claims.